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Jacob Bælum Morten Schostag Nielsen Anders R. Johnsen Carsten Suhr Jacobsen

mRNA/DNA ratios as a measure of bacterial inhibition compared to classical microbial analysis (WP1 and WP2.1). Jacob Bælum Morten Schostag Nielsen Anders R. Johnsen Carsten Suhr Jacobsen. Hypothesis.

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Jacob Bælum Morten Schostag Nielsen Anders R. Johnsen Carsten Suhr Jacobsen

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  1. mRNA/DNA ratios as a measure of bacterial inhibition compared to classical microbial analysis (WP1 and WP2.1) • Jacob Bælum • Morten Schostag Nielsen • Anders R. Johnsen • Carsten Suhr Jacobsen

  2. Hypothesis • Because ammonium oxidation in soil is sensible to changes in environmental conditions it is an ideal microbial process for the use as biomarker for screening of unwanted pesticide effects • mRNA/DNA ratios of the amoA gene can be related to the rate of ammonium oxidation in soil • mRNA/DNA ratios of the amoA gene can be used to measure the effects of pesticides directly in the field without incubation • Ratios between AOB amoA and AOA amoA mRNA might as well be used

  3. Aim • To select and validate a qPCR based assay for quantification of global amoA genes • To select and validate a qPCR based assay for individual quantification of AOB amoA and AOA amoA genes • Investigate if transcription of global amoA genes based on mRNA/DNA ratios or AOB amoA/AOA amoA transcript ratios can be used to determine ammonium oxidation rates in soil • Compare the mRNA based approach to the establised incubation based DS/ISO 14238:1997 method

  4. Nitrogen cycle NH3 NH2OH NO2- amoABC amoABC amoABC • amoA is by far the best characterized • amoA is found both in bacteria (AOB) and archea (AOA) • AOB amoA/AOA amoA expression ratios have been used to study effects of pH on ammonium oxidation

  5. Pilot experiment 5 concentrations 4 sample points for RNA/DNA extraction Pesticide 1+ NH4+ source 3 replicates 100 days of incubation NO3- measurements after 28 and 100 days 5 concentrations 4 sample points for RNA/DNA extraction Pesticide 2 + NH4+ source 3 replicates

  6. Suggestions for choice of pesticide A fungicide - bacause it in general are targeting most unspecific processes A insecticide since this is expected to work the best for the springtails Alf Aagard MST Lise Nistrup ??

  7. DNA/RNA preparation 0.5 g soil snap-shot frozen in liquid N2 CTAB Chloroform/phenol Bead beating PEG precipitation Column cleanup Dnase treatment Hexamer reverse transcription RNA DNA qPCR

  8. amoA qPCR • Suitable PCR primers need to be chosen (default the ones suggested by Prosser) • Numerous primer sets have been published • eventually primer sets for individual amplification of AOB and AOA amoA • Positive controls for standards need to be obtained • ”Real” AOB and AOA strains • Synthetic positive controls in e.coli clones • qPCR optimization

  9. OECD 216 (or DS/ISO 14238:1997?) • SOIL • 0-20 cm depth, ploughlayer • Sieved < 2mm • Sand: 50-75%, • pH. 5.5-7.5. • Soil organic carbon: 0.5-1.5% • Biomass: < 1% of SOC • Storage < 3 months

  10. OECD 216 (or DS/ISO 14238:1997?) • 40-60% WHC • Pesticide added in water or quartz sand • Concentrations: predicted environmental concentration – 5x PEC or geometrical series of 5 concentrations. • Several pesticide concentrations  28 days incubation • N: 5 g/kg alfalfa (ISO: N =100 mg/kg, Alfalfa or (NH3)2SO4) • Cover with perforated polyethylene foil • 20±2° C, dark • Variation between replicates <15% • Agrochemicals: nitrate determination on days 0, 7, 14, 28 and then every 14 days • < 25% effect pesticide non-toxic

  11. Nitrate assay test • Color assay where nitrate is reduced to nitrite. • Test for nitrate-nitrite interference • WHC: 25% • Dried to 8.8% (= 35% of WHC) • Distributed in 10-g portions in 50 ml tubes • Ammonium sulfate added in water (=55% WHC), controls with water only. • Nitrate determination (and DNA-RNA) at days 0, 7, 14, 21, 28.

  12. Mesocosm experiment • Our mRNA/DNA qPCR based method targeting amoA genes will be provided • Mesocosm setup need to be discussed…

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